Alcohol abuse promotes the development of osteoporosis. The prevalence of osteoporosis in alcohol abusers is estimated to be 28-52% compared to approximately 10% prevalence for all adult Americans. Alcohol abuse also suppresses both innate and acquired immune function leading to a higher incidence of infections with increased morbidity and mortality. These apparently unrelated outcomes of alcohol abuse are intricately linked through the effects of alcohol on the cell that is the origin of both osteoclasts and myeloid dendritic cells (mDCs): the osteoclast-dendritic cell (ODC) progenitor. Because of its role in bone maintenance and immune cell production, the ODC is a linchpin of the skeletal and immune systems. This interdependence makes the osteoimmune system particularly vulnerable to the effects of chronic alcohol consumption on the ODC. RANK/c-FOS and Wnt/Frizzled (FZD)/2-catenin pathways are two major signaling systems involved in ODC differentiation to osteoclasts and mDCs, respectively. Integration of these two signaling pathways occurs through NOTCH signaling. NOTCH activation suppresses osteoclastogenesis through inhibiting RANK transcription while promoting FZD (Wnt receptor) gene expression, enhancing dendropoiesis. Chronic alcohol consumption suppressed NOTCH activity in bone marrow cells of rhesus macaques. Furthermore, chronic alcohol consumption increased RANK expression and decreased FZD receptor gene expression through epigenetic mechanisms (RANK promoter CpG hypomethylation and FZD promoter CpG hypermethylation). Little information is available regarding the interplay among these progenitor cell signaling pathways, and the impact of alcohol on these signaling and epigenetic mechanisms remains to be examined. Our hypothesis is that chronic alcohol consumption dysregulates key signaling mechanisms controlling the balance of ODC progenitor lineage differentiation, which leads to enhanced osteoclastogenesis and impaired dendropoiesis. Experiments designed to address three Specific Aims will test the hypotheses that chronic alcohol consumption: (1) promotes osteoclastogenesis through upregulation of RANK signaling;(2) impairs dendropoiesis by inhibiting the FZD/2-catenin signaling pathway;and (3) alters promoter CpG methylation in RANK and FZD genes, further unbalancing ODC progenitor cell differentiation. Genome methylation techniques, as well as lentiviral vector production and the associated protocols will be mastered during the K99 period. These techniques will be employed to complete the proposed research during the R00 period. Grant proposal writing and mentoring duties will complement laboratory research during the R00 phase. The proposed experimental approach will employ a clinically relevant model of chronic alcohol consumption in rhesus macaques and a number of cutting edge technologies. This investigation will also identify key targets for developing therapeutic interventions to treat osteoimmune dysfunction in alcohol abusers. PUBLIC HEALTH RELEVANCE: Alcohol abuse is a critical contributor to both debilitating bone diseases and heightened infection risk. Alcohol- induced dysfunction of bone marrow resident stem cells, which make bone destroying cells and immune cells, is a root cause of these osteoimmune diseases. This project investigates the unifying mechanisms responsible for these serious health problems, which directly increase the medical burden, and will identify therapeutic targets for effectively treating alcoholic patients with osteoporosis.